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EP2999471B1 - Systèmes, procédés et compositions permettant une réticulation - Google Patents

Systèmes, procédés et compositions permettant une réticulation Download PDF

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Publication number
EP2999471B1
EP2999471B1 EP14800815.4A EP14800815A EP2999471B1 EP 2999471 B1 EP2999471 B1 EP 2999471B1 EP 14800815 A EP14800815 A EP 14800815A EP 2999471 B1 EP2999471 B1 EP 2999471B1
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Prior art keywords
cross
riboflavin
illustrates
linking
fluorescence
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EP2999471A4 (fr
EP2999471A1 (fr
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Pavel Kamaev
Marc Friedman
Evan Sherr
David Muller
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Avedro Inc
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Avedro Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/26—Iron; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/12—Ketones
    • A61K31/121—Ketones acyclic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/12—Ketones
    • A61K31/122—Ketones having the oxygen directly attached to a ring, e.g. quinones, vitamin K1, anthralin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/498—Pyrazines or piperazines ortho- and peri-condensed with carbocyclic ring systems, e.g. quinoxaline, phenazine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/525—Isoalloxazines, e.g. riboflavins, vitamin B2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/40—Peroxides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N5/00—Radiation therapy
    • A61N5/06—Radiation therapy using light
    • A61N5/0613—Apparatus adapted for a specific treatment
    • A61N5/062—Photodynamic therapy, i.e. excitation of an agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A61P27/10—Ophthalmic agents for accommodation disorders, e.g. myopia

Definitions

  • the present invention relates generally to treatment of eye disorders, and more particularly, to systems, methods, and compositions that generate cross-linking activity for treatment of eye disorders.
  • a variety of eye disorders such as myopia, keratoconus, and hyperopia, involve abnormal shaping of the cornea.
  • Laser-assisted in-situ keratomileusis (LASIK), for example, is one of a number of corrective treatments that reshape the cornea so that light traveling through the cornea is properly focused onto the retina located in the back of the eye.
  • the success of a particular treatment in addressing abnormal shaping of the cornea depends on the stability of the changes in the corneal structure after the treatment has been applied.
  • treatments may initially achieve desired reshaping of the cornea, the desired effects of reshaping the cornea may be mitigated or reversed at least partially if the collagen fibrils within the cornea continue to change after the desired reshaping has been achieved.
  • some treatments may also initiate cross-linking activity in the corneal tissue.
  • a photosensitizing agent e.g ., riboflavin
  • a cross-linking agent is applied to the cornea as a cross-linking agent.
  • the cross-linking agent is activated by a light source (e.g ., ultraviolet (UV) light) to cause the cross-linking agent to absorb enough energy to cause the release of free oxygen radicals (e.g ., singlet oxygen) and/or other radicals within the cornea.
  • a light source e.g ., ultraviolet (UV) light
  • free oxygen radicals e.g ., singlet oxygen
  • the radicals form covalent bonds between corneal collagen fibrils and thereby cause the corneal collagen fibrils to cross-link and strengthen and stabilize the structure of the cornea.
  • the cross-linking agent may be applied as the primary aspect of some treatments.
  • a cross-linking agent may be applied to treat keratoconus.
  • US 2011/0152219 A1 discloses the use of enhancers "with possibly riboflavin" by the method of corneal crosslinking.
  • the present invention is provided according to claim 1.
  • a composition for applying therapy to a cornea of an eye comprises a cross-linking agent which is riboflavin that generates cross-linking activity in the cornea in response to exposure to a photoactivating light.
  • the composition also comprises at least one additive which is iron.
  • the at least one additive enhances the cross-linking activity generated by the cross-linking agent.
  • the at least one additive may be iron, e.g., provided by FeSO4.
  • a method for applying therapy to a cornea of an eye comprises applying a composition to the cornea, where the composition includes a cross-linking agent that generates cross-linking activity in the cornea in response to exposure to a photoactivating light, and at least one additive different from the cross-linking agent and selected from the group consisting of iron, copper, manganese, chromium, vanadium, aluminum, cobalt, mercury, cadmium, nickel, arsenic, 2,3-butanedione, and folic acid.
  • the method also comprises applying photoactivating light to the cornea to generate cross-linking activity in the cornea.
  • the at least one additive enhances the cross-linking activity generated by the cross-linking agent.
  • the cross-linking agent may be selected from the group consisting of riboflavin, 2,3-butanedione, folic acid, quinoxalines, quinolines, dibucaine, Methotrexate, menadione, and derivatives thereof.
  • the at least one additive may be iron, e.g., provided by FeSO4.
  • the at least one additive may be 2,3-butanedione.
  • the at least one additive may be folic acid.
  • the photoactivating light may be ultraviolet light and/or pulsed (or alternatively continuous). The method may further comprise applying oxygen to the cornea to control the cross-linking activity generated by the cross-linking agent.
  • a method for applying therapy to a cornea of an eye comprises applying a cross-linking agent to the cornea, the cross-linking agent being selected from the group consisting of 2,3-butanedione, folic acid, quinoxalines, quinolines, dibucaine, Methotrexate, menadione, and derivatives thereof.
  • the method also comprises applying photoactivating light to the cornea to generate cross-linking activity in the cornea.
  • the photoactivating light may be ultraviolet light and/or pulsed (or alternatively continuous).
  • the method may further comprise applying oxygen to the cornea to control the cross-linking activity generated by the cross-linking agent.
  • a method for applying therapy to a cornea of an eye comprises applying a hydrogen peroxide to the cornea and applying an iron solution to the cornea after applying the hydrogen peroxide.
  • the hydrogen peroxide and iron solution combine to generate cross-linking activity in the cornea.
  • hydroxyl radicals alone are able to induce protein aggregation. Hydroxyl radicals are considered to be initiators of polymerization not only for proteins. However, a mixture of the hydroxyl radicals with superoxide anions (with excess of the former) stimulates degradation of proteins.
  • Copper ions can be used instead of iron, and it is a promising sign that cross-linking of collagen is observed under this condition.
  • traces of metals such as iron or copper enhances corneal collagen cross-linking with UV light.
  • Other metals that may mediate formation of reactive oxygen and nitrogen species include, for example: manganese, chromium, vanadium, aluminum, cobalt, mercury, cadmium, nickel, or arsenic.
  • De-epithelialized eyes were soaked for 20 minutes with 0.1% riboflavin in dH 2 O only, or 1 mM of FeSO 4 (Iron(II) Sulfate) in 0.1% riboflavin in dH 2 O, in an incubator set at 37°C by using a rubber ring to hold the solution on top of the cornea.
  • Corneas were pan-corneally irradiated with a top hat beam (3% root mean square) for 8 minutes (7.2 J total dose) in a cylinder filled with oxygen with 365-nm light source (pulsing 1 second on, 1 second off) (UV LED NCSU033B[T]; Nichia Co., Tokushima, Japan) at the chosen irradiance (30 mW/cm 2 ) which was measured with a power sensor (model PD-300-UV; Ophir, Inc., Jerusalem, Israel) at the corneal surface. Before the start of irradiation, oxygen's exposure was 2 minutes.
  • Corneal flaps (approximately 200 ⁇ m thick) were excised from the eyes with aid of Intralase femtosecond laser (Abbot Medical Optics, Santa Ana, CA). The average thickness of the corneal flaps was calculated as a difference between the measurements before and after the excision from the eyes with an ultrasonic Pachymeter (DGH Technology, Exton, PA). The flaps were washed with distilled water two times, dried with filter paper, washed with dH 2 O two times, and then dried in a vacuum until the weight change became less than 10% (Rotary vane vacuum pump RV3 A652-01-903, BOC Edwards, West Wales, UK).
  • Each flap was digested for 2.5 hours at 65°C with 2.5 units/ml of papain (from Papaya latex, Sigma) in 1 ml of papain buffer [BBBS (pH 7.0-7.2), 2 mM L-cysteine and 2 mM EDTA].
  • the fluorescence of the papain buffer was taken into account by measuring fluorescence in the absence of tissue and subtracting this value from the fluorescence of the samples.
  • FIG. 3 illustrates the fluorescence of the corneal digests at 450 nm in relation to the non-cross-linked control (F/Fo) with different concentrations of Iron(II) in solution applied during cross-linking.
  • F/Fo non-cross-linked control
  • FIG. 3 illustrates the fluorescence of the corneal digests at 450 nm in relation to the non-cross-linked control (F/Fo) with different concentrations of Iron(II) in solution applied during cross-linking.
  • the presence of Iron(II) in riboflavin solution enhances collagen-related fluorescence at 450 nm after exposure to UVA light, indicative of cross-linking activity.
  • Pig eyes were shipped overnight on ice from an abattoir (SiouxPreme, Sioux City, IA), rinsed in saline. Eyes were several days old at time of experiment. Eyes were cleaned and epithelium was removed. Corneal flaps (approximately 200 ⁇ m thick) were excised from the eyes with aid of Intralase femtosecond laser (Abbot Medical Optics, Santa Ana, CA). The average thickness of the corneal flaps was calculated as a difference between the measurements before and after the excision from the eyes with an ultrasonic Pachymeter (DGH Technology, Exton, PA).
  • Corneal flaps were soaked in either distilled water or diluted H 2 O 2 (1%) for 20 minutes. Flaps soaked in H 2 O 2 were either rinsed twice with distilled water or removed from H 2 O 2 and placed in 0.1% FeSO 4 solution in distilled water for an additional 20 minute soak followed by a 2X rinse with distilled water. Flaps were dried in a vacuum until the weight change became less than 10% (Rotary vane vacuum pump RV3 A652-01-903, BOC Edwards, West Wales, UK).
  • Each flap was digested for 2.5 hours at 65°C with 2.5 units/ml of papain (from Papaya latex, Sigma) in 1 ml of papain buffer [BBBS (pH 7.0-7.2), 2 mM L-cysteine and 2 mM EDTA].
  • the fluorescence of the papain buffer was taken into account by measuring fluorescence in the absence of tissue and subtracting this value from the fluorescence of the samples.
  • FIG. 4 illustrates fluorescence counts of papain digested corneal flaps treated with: (1) dH 2 O; (2) H 2 O 2 ; and (3) H 2 O 2 and 0.1% Iron(II).
  • a fluorescence pattern for the H 2 O 2 + 0.1% Iron(II) condition is similar to normal cross-linking patterns. This demonstrates that cross-linking occurs when flaps are placed in the Iron solution after H 2 O 2 , but not when they were exposed to only H 2 O 2 .
  • Pig eyes were shipped overnight on ice from an abattoir (SiouxPreme, Sioux City, IA), rinsed in saline. Eyes were cleaned and epithelium was removed. Eyes were soaked for 20 minutes with dH 2 O, 0.1% riboflavin in dH 2 O or 0.5 mM FeSO 4 in 0.1% riboflavin in dH 2 O in an incubator set at 37°C by using a rubber ring to hold the solution on top. If specified, eyes were placed in a beaker with a light oxygen stream for 2 minutes in the incubation chamber prior to irradiation.
  • Corneas were pan-corneally irradiated with a top hat beam (3% root mean square) for the chosen time (4 or 8 minutes) with 365-nm light source (UV LED NCSU033B[T]; Nichia Co., Tokushima, Japan) at the chosen irradiance (30 mW/cm 2 , pulsed or non-pulsed) which was measured with a power sensor (model PD-300-UV; Ophir, Inc., Jerusalem, Israel) at the corneal surface. Corneal flaps (approximately 200 ⁇ m thick) were excised from the eyes with aid of Intralase femtosecond laser (Abbot Medical Optics, Santa Ana, CA).
  • the average thickness of the corneal flaps was calculated as a difference between the measurements before and after the excision from the eyes with an ultrasonic Pachymeter (DGH Technology, Exton, PA).
  • the flaps were placed into a biaxial extensometer (CellScale Biotester 5000, Waterloo, ON), using biorake attachments with 5 tines spanning a width of 3 mm. Each sample was stretched at a constant rate of 4 ⁇ m/s in saline at 37°C until sample failure.
  • the flaps were washed with distilled water 2 times, dried with filter paper, washed with dH 2 O two times, and then dried in a vacuum until the weight change became less than 10% (Rotary vane vacuum pump RV3 A652-01-903, BOC Edwards, West Wales, UK).
  • Each flap was digested for 2.5 hours at 65°C with 2.5 units/ml of papain (from Papaya latex, Sigma) in 1 ml of papain buffer [BBBS (pH 7.0-7.2), 2 mM L-cysteine and 2 mM EDTA].
  • FIG. 5 illustrates fluorescence (relative to untreated controls) recorded at 450 nm for: (1) 0.1% riboflavin (continuous wave (CW)); (2) 0.1% riboflavin + 0.5 mM FeSO 4 (CW); (3) 0.1% riboflavin (pulsed wave (PW) + O 2 ); and (4) 0.1% riboflavin + 0.5 mM FeSO 4 (PW + O 2 ).
  • FIG. 6 illustrates average force vs. displacement of each corneal flap measured by tensiometry for: 0.1% riboflavin (CW) (2) and 0.1% riboflavin + 0.5 mM FeSO 4 (CW) (3), relative to controls (1).
  • FIGS. 7-8 illustrate, for repeated experiments, average force vs. displacement of each corneal flap measured by tensiometry for 0.1% riboflavin (PW + O 2 ) (2) and 0.1% riboflavin + 0.5 mM FeSO 4 (PW + O 2 ) (3), relative to controls (1).
  • the relative fluorescence graph of FIG. 5 shows an increase in fluorescence counts when FeSO 4 is added to 0.1% riboflavin as well as when UVA application is changed from a continuous dose to a pulsed dose with oxygen.
  • FIG. 6 shows the tensiometry results from the continuous UVA dose treatment groups.
  • the 0.1% riboflavin group and the 0.1% riboflavin + 0.5 mM FeSO 4 group both display a similar correlation between force and displacement as the displacement increases. Both groups are higher than the control group.
  • FIGS. 7 and 8 show the tensiometry results from the pulsed UVA application with oxygen treatment groups.
  • the 0.1% riboflavin + 0.5 mM FeSO 4 group shows a slightly greater force as the displacement increases.
  • the increase in force was greater for the PW + O 2 treatment groups than the increase in the CW treatment groups.
  • Diacetyl (2,3-butanedione) is an ⁇ -diketone that is present naturally in butter and a variety of foods including dairy products and alcoholic beverages as a product of bacterial fermentation.
  • 2,3-butanedione is a major volatile product detected in the riboflavin solutions after irradiation with UV light.
  • the mechanism includes the interaction between singlet oxygen and riboflavin.
  • FIG. 9 illustrates the mechanism for the formation of 2,3-butanedione from riboflavin and singlet oxygen.
  • the following study conducted an investigation to measure quantitatively the cross-linking efficiency of 2,3-butanedione when using it for corneal cross-linking with and without riboflavin.
  • Pig eyes were shipped overnight on ice from an abattoir (SiouxPreme, Sioux City, IA), rinsed in saline. Eyes were cleaned and epithelium was removed. Eyes were soaked for 20 minutes with 0.1% riboflavin in dH 2 O, or 0.1% 2,3-butanedione (BD) in dH 2 O in an incubator set at 37°C by using a rubber ring to hold the solution on top of the eye.
  • abattoir SiouxPreme, Sioux City, IA
  • Eyes were cleaned and epithelium was removed. Eyes were soaked for 20 minutes with 0.1% riboflavin in dH 2 O, or 0.1% 2,3-butanedione (BD) in dH 2 O in an incubator set at 37°C by using a rubber ring to hold the solution on top of the eye.
  • BD 2,3-butanedione
  • Corneas were pan-corneally irradiated with a top hat beam (3% root mean square) for 4 minutes with 365-nm light source (UV LED NCSU033B[T]; Nichia Co., Tokushima, Japan) at irradiance 30 mW/cm 2 , which was measured with a power sensor (model PD-300-UV; Ophir, Inc., Jerusalem, Israel) at the corneal surface. Corneal flaps (approximately 200 ⁇ m thick) were excised from the eyes with aid of Intralase femtosecond laser (Abbott Medical Optics, Santa Ana, CA).
  • the average thickness of the corneal flaps was calculated as a difference between the measurements before and after the excision from the eyes with an ultrasonic Pachymeter (DGH Technology, Exton, PA).
  • the flaps were then placed into a biaxial extensometer (CellScale Biotester 5000, Waterloo, ON), using biorake attachments with 5 tines spanning a width of 3 mm. Each sample was stretched at a constant rate of 4 ⁇ m/s in saline at 37°C until sample failure.
  • the flaps were washed with distilled water, dried in a vacuum until the weight change became less than 10% (Rotary vane vacuum pump RV3 A652-01-903, BOC Edwards, West Wales, UK).
  • Each flap was digested for 2.5 h at 65°C with 2.5 units/ml of papain (from Papaya latex, Sigma) in 1 ml of papain buffer [BBBS (pH 7.0-7.2), 2 mM L-cysteine and 2 mM EDTA].
  • the fluorescence of the papain buffer was taken into account by measuring fluorescence in the absence of tissue and subtracting this value from the fluorescence of the samples.
  • FIG. 10 illustrates displacement-force diagrams for corneal flaps treated with 1% solution of 2,3-butanedione (BD) in dH 2 O without ultraviolet (UV) light (left panel) and with UV light (right panel) (365 nm, 30 mW for 4 min).
  • BD 2,3-butanedione
  • FIG. 11 illustrates fluorescence (relative to the untreated controls, Fo) recorded at 450 nm from the papain digested 200 ⁇ m-thick corneal flaps, treated with 1% solution of BD with and without UV light.
  • FIG. 12 illustrates displacement-force diagrams for corneal flaps treated with UV light (365 nm, 30 mW for 4 min) and: 0.1% solution of riboflavin in dH 2 O (1); 0.1% BD in dH 2 O (4); and mixture of 0.1% riboflavin and 0.1% BD in dH 2 O (3), relative to controls (1).
  • FIG. 13 illustrates fluorescence (relative to the untreated controls, Fo) recorded at 450 nm from the papain digested 200 ⁇ m-thick corneal flaps, treated with 30 mW UVA for 4 min and: 0.1% solution of riboflavin in dH 2 O; 0.1% BD in dH 2 O; and mixture of 0.1% riboflavin and 0.1% BD in dH 2 O.
  • FIGS. 10 and 11 2,3-butanedione itself (without UV light) does not cross-link corneal flaps, but when irradiated with 365 nm UV light, it leads to the increase of the corneal stiffness and fluorescence output recorded from the treated cornea.
  • FIGS. 12 and 13 show change in the stiffness of the corneal flaps when mixture of BD with riboflavin is used for the cross-linking.
  • 2,3-butanedione can be used as an additive to a riboflavin formulation to increase cross-linking efficacy.
  • 2,3-butanedione can also be used as a primary cross-linking agent (without riboflavin).
  • Riboflavin is hydrolyzed in alkaline solution to give urea and 1,2-dihydro-6,7-dimethyl-2-keto-1-D-ribityl-3-quinoxaline-carboxylic acid among the hydrolysis products.
  • FIG. 24 illustrates alkaline hydrolysis of riboflavin (A) into 1,2-dihydro-6,7-dimethyl-2-keto-1-D-ribityl-3-quinoxaline-carboxylic acid (B).
  • FIG. 25 illustrates UV/Visible (Vis) light spectra of the 0.1% riboflavin-5-phosphate in BBBS kept at 120 °C for different amounts of time.
  • FIG. 25 illustrates concentration of riboflavin decreases with time ( FIG. 25 , see absorbance change at 450 nm).
  • the rate of destruction of riboflavin depends also on its concentration in solution.
  • the present inventors have found that a 0.1% solution hydrolyzes 1.3 times more quickly than a 0.5% solution (as shown in FIG 26 ).
  • FIG. 26 illustrates the rate of hydrolysis of riboflavin at 120 °C in BBBS as measured by absorbance at 450 nm (0.1% solution and 0.5% solution, A 0 is the absorbance before heating).
  • FIG. 27 illustrates UV/Vis spectra which is obtained from FIG. 25 by subtracting absorbance of the remaining riboflavin.
  • FIG. 28 illustrates spectral analysis of the hydrolyzed solution after 90 min at 120°C (absorbance of residual riboflavin was subtracted from the analyzed spectrum).
  • FIG. 29 illustrates change in the absorbance of the different peaks during the time of hydrolysis.
  • the sodium salt of 1,2-Dihydro-6,7-dimethyl-2-keto-1-D-ribityl-3-quinoxalinecarboxylic acid monohydrate 2 an early stage alkaline degradation product of riboflavin, as shown in FIG. 30 , can be synthesized.
  • the quinoxaline 2 can be prepared by using a synthetic protocol from Surrey et al. J. Am. Chem. Soc. 1951, 73, 3236-2338 .
  • FIG. 31 illustrates NMR spectrum of the synthesized riboflavin degradation product 2.
  • FIG. 32 illustrates monophosphorylated riboflavin (5-FMN) in buffered blood bank saline without thermal treatment.
  • FIG. 33 illustrates 5-FMN in buffered blood bank saline after 1 hour of thermal treatment.
  • FIG. 34 illustrates 5-FMN in buffered blood bank saline after 2 hours of thermal treatment.
  • FIG. 35 illustrates 5-FMN in buffered blood bank saline after 3 hours of thermal treatment.
  • FIG. 36 illustrates 5-FMN in buffered blood bank saline after 4 hours of thermal treatment.
  • FIG. 37 illustrates HPLC trace of the synthesized riboflavin degradation product 2.
  • a second degradation product A is also produced during the thermal degradation of 5-FMN. Due to its more polar characteristics (shorter retention time) it is assumed that this peak corresponds to the phosphorylated quinoxaline compound. This assumption can be confirmed by comparing the UV spectra of both compounds. The similarity of the spectra indicates that no change at the chromophore has taken place. Therefore, it is assumed that this quinoxaline intermediate is formed by the loss of one molecule of urea without the hydrolysis of the phosphorous ester.
  • FIG. 38 illustrates absorption spectra of thermally heated (red line) and not heated (blue line) riboflavin solutions (recorded in a quartz cuvette with 200 ⁇ m optical path).
  • FIG. 39 illustrates the Difference between two spectra in FIG. 32 .
  • FIG. 40 Fluorescence of the digested with papain buffer corneas are presented on FIG. 40 .
  • FIG. 40 illustrates fluorescence of the digested corneas: non-cross-linked control (black line), cross-linked with 0.1% riboflavin which was not heated (blue line), cross-linked with thermally treated riboflavin solution (red line).
  • FIG. 41 illustrates relative fluorescence of the cross-linked corneal samples: red - using thermally treated riboflavin solution, blue - using not heated 0.1% riboflavin solution.
  • FIG. 42 Sodium salt of 1,2-Dihydro-6,7-dimethyl-2-keto-1-D-ribityl-3-quinoxalinecarboxylic acid monohydrate, as shown in FIG. 42 , has a strong absorbance at 360 nm (as shown in FIGS. 43 and 44 ) and noticeable fluorescence with a maximum around 460 nm (as shown in FIG. 45 ).
  • FIG. 43 illustrates absorbance spectrum of 0.001% solution of sodium salt of 1,2-Dihydro-6,7-dimethyl-2-keto-1-D-ribityl-3-quinoxalinecarboxylic acid monohydrate in BBBS (quartz, 1 cm light path)
  • FIG. 43 illustrates absorbance spectrum of 0.001% solution of sodium salt of 1,2-Dihydro-6,7-dimethyl-2-keto-1-D-ribityl-3-quinoxalinecarboxylic acid monohydrate in BBBS (quartz, 1 cm light path)
  • FIG. 43 illustrates absorbance spectrum of 0.001%
  • FIG. 44 illustrates UV absorbance of sodium salt of 1,2-Dihydro-6,7-dimethyl-2-keto-1-D-ribityl-3-quinoxalinecarboxylic acid monohydrate at 360 nm (solution in BBBS, quartz cuvette with 1 cm light path).
  • FIG. 45 illustrates fluorescence of sodium salt of 1,2-Dihydro-6,7-dimethyl-2-keto-1-D-ribityl-3-quinoxalinecarboxylic acid monohydrate in BBBS solutions (excitation 360 nm).
  • FIG. 46 illustrates fluorescence of the digested corneas: non-cross-linked control (black line), cross-linked with 0.1% riboflavin in BBBS (red line), cross-linked with 0.1% sodium salt of 1,2-Dihydro-6,7-dimethyl-2-keto-1-D-ribityl-3-quinoxalinecarboxylic acid monohydrate (blue line).
  • FIG. 47 illustrates fluorescence of the digested corneal flaps: non-cross-linked control (black line), cross-linked with 0.1% riboflavin in BBBS (red line), cross-linked with 0.1% sodium salt of 1,2-Dihydro-6,7-dimethyl-2-keto-1-D-ribityl-3-quinoxalinecarboxylic acid monohydrate (blue line).
  • Fluorescence of the corneal flaps which were cross-linked with sodium salt of 1,2-Dihydro-6,7-dimethyl-2-keto-1-D-ribityl-3-quinoxalinecarboxylic acid monohydrate was higher than the fluorescence of the corneal flaps cross-linked with riboflavin. This suggests a lower sensitivity to the oxygen presence when sodium salt of 1,2-Dihydro-6,7-dimethyl-2-keto-1-D-ribityl-3-quinoxalinecarboxylic acid monohydrate is used as a cross-linking agent.
  • this salt may be employed in treatments where lower sensitivity to the presence of oxygen is advantageous.
  • FIG. 48 0.17% and 0.017% solutions of 3-hydroxy-2-quinoxalinecarboxylic acid, as shown in FIG. 48 , were prepared in BBBS and recorded absorbance (in 200 ⁇ m and 1 cm - thick quartz quvettes) as presented in the FIG. 49.
  • FIG. 49 illustrates absorbance spectra of 3-hydroxy-2-quinoxalinecarboxylic acid.
  • FIG. 50 illustrates fluorescence of 3-hydroxy-2-quinoxalinecarboxylic acid's solutions with different concentrations in BBBS, recorded with excitation of 360 nm.
  • FIG. 51 illustrates fluorescence of the papain digested corneal flaps (200 ⁇ m thick) cross-linked with 0.17% (red lines) and 0.017% (blue lines) solutions of 3-hydroxy-2-quinoxalinecarboxylic acid in BBBS (no epithelium, 20 min soaking time, 30 mW/cm 2 for 4 min), relative to non-cross-linked controls (black lines).
  • BBBS no epithelium, 20 min soaking time, 30 mW/cm 2 for 4 min
  • FIG. 52 illustrates tensiometry plots of 200- ⁇ m thick corneal flaps irradiated at 30 mW/cm2 for 4 min, preliminary saturated for 20 min with 0.17% riboflavin (red lines) and 0.17% 3-hydroxy-2-quinoxalinecarboxylic acid (3H2QXCA, green lines)relative to non-cross-linked controls (black lines).
  • FIG. 52 illustrates tensiometry plots of 200- ⁇ m thick corneal flaps irradiated at 30 mW/cm2 for 4 min, preliminary saturated for 20 min with 0.17% riboflavin (red lines) and 0.17% 3-hydroxy-2-quinoxalinecarboxylic acid (3H2QXCA, green lines)relative to non-cross-linked controls (black lines).
  • FIG. 53 illustrates relative fluorescence of the papain digested corneal flaps (200 ⁇ m thick) cross-linked with 0.1% riboflavin (blue bar, solution 2) and 0.1% riboflavin containing 0.02% solution of 3-hydroxy-2-quinoxalinecarboxylic acid in BBBS (red bar, solution 1) (no epithelium, 20 min soaking time, 30 mW/cm 2 for 4 min), where F 0 - fluorescence of a non-cross-linked flap.
  • FIG. 54 4-methyl-3-oxo-3,4-dihydro-2-quinoxaline carboxylic acid as shown in FIG. 54 has a strong absorbance at 360 nm (as shown in FIG. 55 ) and some fluorescence with a maximum around 460 nm (as shown in FIG. 56 ).
  • FIG. 55 illustrates absorbance spectrum of 0.01 mg/ml solution of 4-methyl-3-oxo-3,4-dihydro-2-quinoxaline carboxylic acid in BBBS (quartz, 1 cm light path).
  • FIG. 56 illustrates fluorescence of 4-methyl-3-oxo-3,4-dihydro-2-quinoxaline carboxylic acid in BBBS solutions (excitation 360 nm).
  • FIG. 55 illustrates absorbance spectrum of 0.01 mg/ml solution of 4-methyl-3-oxo-3,4-dihydro-2-quinoxaline carboxylic acid in BBBS (quartz, 1 cm light path).
  • FIG. 56 illustrates fluorescence of
  • 57 illustrates fluorescence of the papain-digested corneal flaps: non-cross-linked control (black line), cross-linked with 0.1 mg/ml (red line) and 1 mg/ml (green line) solutions of 4-methyl-3-oxo-3,4-dihydro-2-quinoxaline carboxylic acid in BBBS, where corneas were de-epithelialized, soaked with the solution of the cross-linker for 20 min and then irradiated for 4 min with 30 mW/cm 2 UVA light (360 nm).
  • systems and methods for treating the eye employ cross-linking agents that are produced during the hydrolysis of riboflavin.
  • hydrolysis of riboflavin produces sodium salt of 1,2-Dihydro-6,7-dimethyl-2-keto-1-D-ribityl-3-quinoxalinecarboxylic acid monohydrate. It has been discovered that this salt is less susceptible to oxygen starvation ( i.e., lower sensitivity to oxygen presence) than conventional treatment solutions of riboflavin. When this salt is used alone or in combination with solutions of riboflavin, more effective cross-linking can be achieved for eye treatments.
  • systems and methods employ thermal treatment of riboflavin solutions to achieve the results of the hydrolysis of riboflavin, including the production of cross-linking agents.
  • systems and methods employ quinoxalines to cause cross-linking activity for eye treatments.
  • Quinoxaline also called benzopyrazine
  • Quinoxaline derivatives are widely distributed in nature and many of them, such as the antibiotics, echinomycin, levomycin and actinoleutin possess very useful biological activity.
  • a large number of synthetic quinoxalines have also shown antibacterial, fungicidal, insecticidal, anticancer, tranquilizing and antidepressant properties.
  • the ability of the quinoxalines to act as cross-linking agents may be employed with these other benefits, e.g., antibacterial or fungicidal characteristics, for certain eye treatments.
  • the collaborative work by synthetic and screening research groups have continuously been carried out to create various biologically active quinoxalines.
  • quinoxaline 1,4-dioxides have been shown antibacterial and quinoxaline-2,3-dithione cyclic dithio-carbonate (Morestan) and trithiocarbonate (Eradox) (as shown in FIG. 59 ) possess fungicidal and insecticidal effects.
  • the 2,3,7-trichloro-6-methylsulfamoyl quinoxaline has been patented as anticancer agent.
  • 2-Phenyl-3-piperidino quinoxaline and some of its derivatives are selective herbicides.
  • Olaquindox N-(2-hydroxyethyl)-3-methyl-2-quinoxalinecarboxamide 1,4-dioxide) (VETRANALTM) has been used since 1975 as a growth promoter for farm animals because it has antibacterial characteristics. Olaquindox is related to the general class of quinoxalines. A commercially available preparation contains 10% olaquindox as an active ingredient in a calcium carbonate carrier together with 1.5% glyceryl polyethyleneglycol ricinoleate to reduce dust during preparation. The concentration of olaquindox in the feed is generally 50 ppm.
  • olaquindox Irradiated in the presence of human serum albumin, olaquindox disappears completely within 20 seconds; the N-monoxides are formed and a modified albumin which has altered properties in isoelectric focussing and electrophoresis systems.
  • Pig eyes were shipped overnight on ice from an abattoir (SiouxPreme, Sioux City, IA), rinsed in saline. Eyes were cleaned and epithelium was removed. Eyes were soaked for 20 minutes with 0.4% Olaquindox in PBS in an incubator set at 37°C by using a rubber ring to hold the solution on top. Some eyes were irradiated on air with continuous UVA light, and some placed in a beaker with a light oxygen stream for 2 minutes in the incubation chamber prior to irradiation.
  • Corneas were pan-corneally irradiated with a top hat beam (3% root mean square) for 4 or 8 minutes with 365-nm light source (UV LED NCSU033B[T]; Nichia Co., Tokushima, Japan) at the chosen irradiance (30 mW/cm2, pulsed 1 sec on 1 sec off). UV irradiance was measured with a power sensor (model PD-300-UV; Ophir, Inc., Jerusalem, Israel) at the corneal surface. Corneal flaps (approximately 200 ⁇ m thick) were excised from the eyes with aid of Intralase femtosecond laser (Abbot Medical Optics, Santa Ana, CA).
  • the average thickness of the corneal flaps was calculated as a difference between the measurements before and after the excision from the eyes with an ultrasonic Pachymeter (DGH Technology, Exton, PA.
  • the flaps were placed into a biaxial extensometer (CellScale Biotester5000, Waterloo, ON), using biorake attachments with 5 tines spanning a width of 3 mm. Each sample was stretched at a constant rate of 4 ⁇ m/s in saline at 37°C until sample failure.
  • the flaps were washed with distilled water 2 times, dried with filter paper, washed with dH 2 O two times, and then dried in a vacuum until the weight change became less than 10% (Rotary vane vacuum pump RV3 A652-01-903, BOC Edwards, West Wales, UK).
  • Each flap was digested for 2.5 hours at 65°C with 2.5 units/ml of papain (from Papaya latex, Sigma) in 1 mL of papain buffer [BBBS (pH 7.0-7.2), 2 mM L-cysteine and 2 mM EDTA].
  • FIG. 22 illustrates biaxial extensiometry of 200 um thick corneal flaps, soaked with 0.4% Olaquindox in PBS with irradiation for 4 min with 30 mW/cm2 (CW) (2) and irradiation for 8 minutes (1 second on:1 second off) with 30 mW/cm2 of pulsed UVA light and O 2 (3), relative to controls (1).
  • FIG. 23 illustrates relative fluorescence recorded at 450 nm of the cross-linked flaps with 0.4% Olaquindox in PBS: (1) non-irradiated control; (2) irradiation for 4 min with 30 mW/cm 2 continuously (CW); and (3): irradiation for 8 minutes (1 second on:1 second off) with 30 mW/cm2 of pulsed UVA light and O 2 .
  • Olaquindox is an effective water-soluble cross-linking agent.
  • FA Folic acid
  • PCA pterine-6-carboxylic acid
  • PCA is an efficient photosensitizer and generator of singlet oxygen. Therefore, both FA and PCA can generate collagen cross-linking.
  • FA may be used in combination with riboflavin because addition of riboflavin markedly intensifies oxidation of FA while most of the riboflavin remains undecomposed.
  • FA is soluble in water with dependence on pH and temperature.
  • solubility in a phosphate buffer was 5.5 mg/ml at 25°C and pH of the final solution was 7.0.
  • FA has UV light absorbance at 400 nm and below (the long wave peak at 360 nm as shown in FIG. 16 ), and fluorescence with maximum around 460 nm (as shown in FIG 17 ).
  • FIG. 18 shows the absorbance of FA at 360 nm as a function of FA concentration in phosphate buffer.
  • Pig eyes were shipped overnight on ice from an abattoir (SiouxPreme, Sioux City, IA), rinsed in saline. The eyes were cleaned and epithelium was removed. Sodium Phosphate Buffer (pH 7.6, made with Sodium Phosphate Monobasic, Sodium Phosphate Dibasic and Sodium Chloride in distilled water) was used as the buffer for all solutions. The final pH values for riboflavin solution, FA solution, and their mixture were in the range of 7.3-7.4. The eyes were soaked for 20 minutes with 0.1% FA, 0.1% Riboflavin, or 0.1% FA + 0.1% riboflavin in an incubator set at 37°C by using a rubber ring to hold the solution on top.
  • the eyes were placed in a beaker filled with pure oxygen for 2 minutes in the incubation chamber prior to irradiation.
  • Corneas were pan-corneally irradiated with a top hat beam (3% root mean square) for 8 minutes with 365-nm light source (UV LED NCSU033B[T]; Nichia Co., Tokushima, Japan) at the chosen irradiance (30 mW/cm 2 , pulsed 1 second on: 1 second off) which was measured with a power sensor (model PD-300-UV; Ophir, Inc., Jerusalem, Israel) at the corneal surface.
  • a power sensor model PD-300-UV; Ophir, Inc., Jerusalem, Israel
  • Corneal flaps (approximately 200 ⁇ m thick) were excised from the eyes with aid of Intralase femtosecond laser (Abbot Medical Optics, Santa Ana, CA). The average thickness of the corneal flaps was calculated as a difference between the measurements before and after the excision from the eyes with an ultrasonic Pachymeter (DGH Technology, Exton, PA). The flaps were placed into a biaxial extensometer (CellScale Biotester 5000, Waterloo, ON), using biorake attachments with 5 tines spanning a width of 3 mm. Each sample was stretched at a constant rate of 4 ⁇ m/s in saline at 37°C until sample failure.
  • the flaps were washed with distilled water 2 times, dried with filter paper, washed with dH 2 O two times, and then dried in a vacuum until the weight change became less than 10% (Rotary vane vacuum pump RV3 A652-01-903, BOC Edwards, West Wales, UK).
  • Each flap was digested for 2.5 hours at 65°C with 2.5 units/ml of papain (from Papaya latex, Sigma) in 1 ml of papain buffer [BBBS (pH 7.0-7.2), 2 mM L-cysteine and 2 mM EDTA].
  • FIG. 19 illustrates displacement vs. force curves for corneal samples: (1) not exposed to UV light; (2) 0.1% riboflavin, exposed to UV light; (3) 0.1% FA, exposed to UV light; and (4) mixture of 0.1% riboflavin and 0.1% FA, exposed to UV light, with UV exposure of 365 nm, 30 mW/cm 2 , pulsed 1 second on: 1 second off for 8 minutes total, with oxygen ambience over the cornea.
  • FIG. 20 illustrates fluorescence of the corneal samples after digestion with papain (excitation 360 nm): (1) not exposed to UV light; (2) 0.1% riboflavin, exposed to UV light; (3) 0.1% FA, exposed to UV light; and (4) mixture of 0.1% riboflavin and 0.1% FA, exposed to UV light, with UV exposure at 365 nm, 30 mW/cm 2 , pulsed 1 second on: 1 second off for 8 minutes total, with oxygen ambience over the cornea.
  • papain excitation 360 nm
  • FIG. 19 the effect of the exposure of UV light on corneal collagen is very similar for all three groups of the studied solutions (0.1% riboflavin, 0.1% FA, and mixture of 0.1% riboflavin with 0.1% FA). As compared to the unexposed controls, soaking with a solution and then exposing it to UV leads to significant stiffening of a corneal sample.
  • FIG. 20 shows that fluorescence of the cross-linked collagen samples increases as compared to the non-exposed to UV control samples.
  • FA can also be used as a primary cross-linking agent (without riboflavin).
  • FIG. 21 illustrates displacement vs. force curves for corneal samples (thickness 300 um, 3 samples in each group): (1) controls unexposed to UV light; (2) 0.1% FA in a buffer, exposed UV light; (3) 0.1% riboflavin in buffer saline solution, exposed to UV light; and (4) mixture of 0.1% FA in 0.1% riboflavin in buffer saline solution, exposed to UV light, UV exposure at 365 nm, 30 mW/cm 2 , pulsed 1 second on: 1 second off for 8 minutes total, with oxygen ambience over the cornea.
  • chloroquine, hydroxychloroquine, quinine, and dibucaine may possess photosensitizing capability in aqueous solutions, e.g., by irradiation with 365 nm UV light.
  • Chloroquine, hydroxychloroquine, and quinine are related to the general class of quinolines. According to aspects of the present disclosure, these drugs, based on their photosensitizing capability, may be applied as cross-linking agents in treatments of the cornea.
  • Methotrexate is a generic name of an immunosuppressive medication which has been used for treatment of certain cancers, and inflammatory diseases such as rheumatoid arthritis and uveitis.
  • MTX has a significant UV-light absorbance at 360 nm.
  • An idea of using MTX as a possible collagen cross-linker comes from the data about MTX photosensitizing properties measured on rabbit eyes conjunctive.
  • UVA-photosensitization of thymidine has been observed with menadione (vitamin K 3 ), as shown in FIG. 61 , and formation of the photo-oxidation product 5,6-dihydroxy-5,6-dihydrothymidine occurs after single electron transfer reactions between triplet state menadione and thymidine.
  • Menadione can be a UVA photosensitizer, and therefore, a cross-linking agent for collagen.
  • riboflavin is combined with Iron(II) to enhance the cross-linking activity generated by the riboflavin.
  • cross-linking treatments employ an Iron(II) solution in combination with a hydrogen peroxide pre-soak.
  • 2,3-butanedione is employed to increase the efficacy of corneal cross-linking with a photosensitizer, such as riboflavin.
  • folic acid is employed in combination with a photosensitizer, such as riboflavin, to enhance cross-linking activity.
  • a photosensitizer such as riboflavin
  • 2,3-butanedione, folic acid, a quinoxaline, a quinoline, dibucaine, Methotrexate, menadione, or a derivative thereof is applied as a cross-linking agent.
  • FIG. 62 provides a block diagram of an example delivery system 100 for delivering cross-linking agent(s) 130 (optionally combined with additive(s) 140) and photo-activating light to a cornea 2 of an eye 1 in order to initiate cross-linking of corneal collagen within the cornea 2.
  • the delivery system 100 includes one or more applicators 132 for applying the cross-linking agent(s) 130 to the cornea 2.
  • the cross-linking agent(s) 130 may include any of the photosensitizers described above.
  • the cross-linking agent(s) 130 may be combined with any of the additives 140 described above to enhance the cross-linking activity generated by the cross-inking agent.
  • the additives described above can as primary cross-linking agents, and vice versa.
  • the one or more applicators 132 may include an eye dropper, syringe, or the like for applying the cross-linking agent(s) 130 in a solution.
  • the one or more applicators 132 may apply the cross-linking agent(s) 130 according to a particular pattern on the cornea 2 where cross-linking activity may be more advantageous.
  • the delivery system 100 may also deliver oxygen 150 to the cornea to control the cross-linking activity further.
  • the delivery system 100 includes a light source 110 and optical elements 112 for directing the photo-activating light to the cornea 2.
  • the optical elements 112 may include, for example, one or more mirrors or lenses for directing and focusing the photo-activating light emitted by the light source 110 according to a particular pattern on the cornea 2 suitable for activating the cross-linking agent(s) 130.
  • the light source 110 may be an ultraviolet (UV) light source, and the photo-activating light directed to the cornea 2 through the optical elements 112 activates the cross-linking agent(s) 130.
  • the light source 110 may also alternatively or additionally emit photons with greater or lesser energy levels than UV light photons.
  • the optical elements 112 can be used to focus the light emitted by the light source 110 to a particular focal plane within the cornea 2, such as a focal plane that includes a mid-depth region 2B.
  • the optical elements 112 may include one or more beam splitters for dividing a beam of light emitted by the light source 110, and may include one or more heat sinks for absorbing light emitted by the light source 110.
  • the optical elements 112 may further include filters for partially blocking wavelengths of light emitted by the light source 110 and for advantageously selecting particular wavelengths of light to be directed to the cornea 2 for activating the cross-linking agent(s) 130.
  • the delivery system 100 also includes a controller 120 that may be coupled to the one or more applicators 132, the light source 110, and/or the optical elements 112.
  • the controller 120 can control the regions of the cornea 2 that receive the cross-linking agent(s) 130 and/or that are exposed to the light source 110.
  • the controller 120 can control the particular regions of the cornea 2 that are strengthened and stabilized through cross-linking of the corneal collagen fibrils.
  • the cross-linking agent(s) 130 can be applied generally to the eye 1, without regard to a particular region of the cornea 2 requiring strengthening, but the light source 110 can be selectively directed to particular regions of the cornea 2 requiring strengthening, and thereby control the region of the cornea 2 wherein cross-linking is initiated by controlling the regions of the cornea 2 that are exposed to the light source 110.
  • the controller 120 may control any combination of: wavelength, bandwidth, intensity, power, location, depth of penetration, and duration of treatment.
  • the controller 120 may include hardware and/or software elements, and may be a computing device.
  • the controller 120 may include a processor, memory storage, a microcontroller, digital logic elements, software running on a computer processor, or any combination thereof.
  • the controller 120 may be replaced by two or more separate controllers or processors.
  • the function of the controller 120 can be partially or wholly replaced by a manual operation.
  • the applicator 132 can be manually operated to deliver the cross-linking agent(s) 130 to the cornea 2 without the assistance of the controller 120.
  • the controller 120 can operate the applicator 132 and/or the optical elements 112 according to inputs dynamically supplied by an operator of the delivery system 100 in real time, or can operate according to a pre-programmed sequence or routine.

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Claims (3)

  1. Composition à utiliser pour appliquer une thérapie de réticulation améliorée à une cornée d'un œil, comprenant :
    un agent de réticulation qui est la riboflavine, l'agent de réticulation générant une activité de réticulation dans la cornée en réponse à une exposition à une lumière photo-activatrice ; et
    un additif différent de l'agent de réticulation et comprenant du fer pour améliorer l'activité de réticulation générée par l'agent de réticulation.
  2. Composition à utiliser selon la revendication 1, dans laquelle le fer est fourni par FeSO4.
  3. Composition à utiliser selon la revendication 1, dans laquelle l'additif améliore l'activité de réticulation générée par la riboflavine en accélérant la décomposition de peroxyde d'hydrogène pendant une photo-activation de la riboflavine et en augmentant une concentration de radicaux hydroxyles.
EP14800815.4A 2013-05-19 2014-05-19 Systèmes, procédés et compositions permettant une réticulation Active EP2999471B1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4480461A3 (fr) 2010-03-19 2025-02-12 Avedro, Inc. Systèmes d'application et de surveillance de thérapie oculaire
US9622911B2 (en) 2010-09-30 2017-04-18 Cxl Ophthalmics, Llc Ophthalmic treatment device, system, and method of use
US9020580B2 (en) 2011-06-02 2015-04-28 Avedro, Inc. Systems and methods for monitoring time based photo active agent delivery or photo active marker presence
EP2830627B1 (fr) 2012-03-29 2024-05-01 Epion Therapeutics, Inc. Solutions de traitement oculaire, dispositifs d'administration et procédés améliorant l'administration
WO2013149075A1 (fr) 2012-03-29 2013-10-03 Cxl Ophthalmics, Llc Compositions et procédés de traitement ou de prévention de maladies associées au stress oxydatif
KR102416876B1 (ko) 2014-10-27 2022-07-05 아베드로 인코퍼레이티드 눈의 교차-결합 처리를 위한 시스템 및 방법
WO2016077747A1 (fr) 2014-11-13 2016-05-19 Avedro, Inc. Étalon de réseau à commande de phase à représentation virtuelle multipasse
US20160175442A1 (en) * 2014-12-02 2016-06-23 Avedro, Inc. Systems, Methods, and Compositions For Cross-Linking Treatments of an Eye
EP3285704B1 (fr) 2015-04-24 2020-11-18 Avedro Inc. Systèmes pour photoactiver un photosensibilisant appliqué à un oeil
WO2016191342A1 (fr) 2015-05-22 2016-12-01 Avedro, Inc. Systèmes et procédés de surveillance de l'activité de réticulation pour des traitements de la cornée
KR102706856B1 (ko) * 2015-07-21 2024-09-19 아베드로 인코퍼레이티드 광증감제로 눈을 치료하기 위한 시스템 및 방법
WO2017180851A1 (fr) 2016-04-13 2017-10-19 Avedro, Inc. Systèmes et procédés d'administration de médicaments à un œil
JP7324208B2 (ja) * 2018-01-05 2023-08-09 ユニバーシティー オブ ユタ リサーチ ファウンデーション 近視進行の治療
US10932864B2 (en) 2018-11-28 2021-03-02 Rxsight, Inc. Tracking-based illumination control system
US11013593B2 (en) 2018-12-02 2021-05-25 Rxsight, Inc. Light adjustable lens tracking system and method
JP7515498B2 (ja) * 2018-12-04 2024-07-12 アヴェドロ インコーポレイテッド 眼の治療のための化合物および組成物
CN113811527B (zh) 2019-01-25 2024-12-31 艾弗德罗股份有限公司 双(双吖丙啶)衍生物作为光交联剂治疗角膜扩张性疾病
KR102234258B1 (ko) * 2019-01-28 2021-03-31 경북대학교 산학협력단 안압이 상승된 동물 모델, 그의 제조방법 및 그를 이용한 녹내장 치료제의 스크리닝 방법

Family Cites Families (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1022373C (zh) * 1989-10-11 1993-10-13 刘长久 动物营养液
US5219895A (en) * 1991-01-29 1993-06-15 Autogenesis Technologies, Inc. Collagen-based adhesives and sealants and methods of preparation and use thereof
US5965620A (en) * 1993-07-23 1999-10-12 Vide Pharmaceuticals Methods and compositions for ATP-sensitive K+ channel inhibition for lowering intraocular pressure
FR2849383B1 (fr) * 2002-12-26 2005-09-30 Jean Noel Thorel Composition trophique en milieu aqueux, et ses applications, notamment en ophtalmologie
EP1740168A2 (fr) * 2004-04-21 2007-01-10 Advanced Ocular Systems Limited Antiprostaglandines pour le traitement de pathologies oculaires
JP5894364B2 (ja) * 2007-08-16 2016-03-30 ザ スキーペンズ アイ リサーチ インスティチュート インコーポレイテッド 眼および付属器組織の炎症を処置するための治療組成物
MX2010009923A (es) * 2008-03-14 2010-12-17 Euclid Systems Corp Irradiacion ultravioleta para tratar trastornos de debilidad en la cornea.
IT1393402B1 (it) * 2008-08-28 2012-04-20 Sooft Italia Spa Uso di enhancer eventualmente con riboflavina, nonche' relative composizioni oftalmiche per cross-linking corneale del cheratocono o di altre patologie ectasiche corneali
US20100159029A1 (en) * 2008-12-23 2010-06-24 Alcon Research, Ltd. Composition and nutritional supplements for improving ocular health and reducing ocular inflammatory response
US8299079B2 (en) * 2009-05-22 2012-10-30 Kaufman Herbert E Preparations and methods for ameliorating or reducing presbyopia
US20120289886A1 (en) * 2010-04-13 2012-11-15 Avedro, Inc. Controlled application of cross-linking agent
US20140113009A1 (en) * 2012-10-24 2014-04-24 Avedro, Inc. Controlled application of cross-linking agent

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
None *

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WO2014189849A1 (fr) 2014-11-27
US20140343480A1 (en) 2014-11-20
EP3858356A2 (fr) 2021-08-04
EP3858356A3 (fr) 2021-12-01
EP2999471A4 (fr) 2017-01-25
EP2999471A1 (fr) 2016-03-30

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